Engineered microorganism producing homo-succinic acid and method for preparing succinic acid using the same

ABSTRACT

The present invention relates to a mutant microorganism, which is selected from the group consisting of genus  Mannheimia , genus  Actinobacillus  and genus  Anaerobiospirillum , producing homo-succinic acid and a method for producing homo-succinic acid using the same, and more particularly to a mutant microorganism producing succinic acid at a high concentration while producing little or no other organic acids in anaerobic conditions, which is obtained by disrupting a gene encoding lactate dehydrogenase (ldhA), a gene encoding phosphotransacetylase (pta), and a gene encoding acetate kinase (ackA), without disrupting a gene encoding pyruvate formate lyase (pfl), as well as a method for producing succinic acid using the same. The inventive mutant microorganism has the property of having a high growth rate and succinic acid productivity while producing little or no organic acids, as compared to the prior strains producing succinic acid. Thus, the inventive mutant microorganism is useful to produce succinic acid for industrial use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. §111(a) andis a continuation-in-part of International Patent Application No.PCT/KR2007/003574 filed on 25 Jul. 2007 entitled “Novel EngineeredMicroorganism Producing Homo-Succinic Acid and Method for PreparingSuccinic Acid Using the Same” in the name of Sang Yup LEE, et al., whichclaims priority of Korean Patent Application No. 10-2006-0071666 filedon 28 Jul. 2006, both of which are hereby incorporated by referenceherein in their entirety.

TECHNICAL FIELD

The present invention relates to a mutant microorganism, selected fromthe group consisting of genus Mannheimia, genus Actinobacillus and genusAnaerobiospirillum, producing homo-succinic acid and a method forpreparing homo-succinic acid using the same, and more particularly to amutant microorganism, which is selected from the group consisting ofgenus Mannheimia, genus Actinobacillus and genus Anaerobiospirillum,producing succinic acid at high concentration while producing little orno other organic acids in anaerobic conditions, which is obtained bydisrupting a gene encoding lactate dehydrogenase (ldhA), a gene encodingphosphotransacetylase (pta), and a gene encoding acetate kinase (ackA),without disrupting a gene encoding pyruvate formate lyase (pfl), as wellas a method for preparing succinic acids using the same.

BACKGROUND ART

Succinic acid (HOOCCH₂CH₂COOH), a dicarboxylic acid consisting of 4carbons, is an organic acid having high utilities, which is widely usedas a precursor of medicine, food, cosmetics, and chemical products ofother industries (Zeikus et al, Appl. Microbiol. Biotechnol., 51:545,1999; Song et al, Enzyme Microbial Technol., 39:352, 2006).Particularly, the demand for succinic acid is expected to bedramatically increased as a main source of biodegradable macromolecules,with the latest sharp increase in petroleum prices (Willke et al, Appl.Microbiol. Biotechnol., 66:131, 2004).

Succinic acid can be produced by chemical synthesis and fermentation.However, most of succinic acid for industrial use is currently producedthrough chemical synthesis methods using n-butane and acetylene derivedfrom petroleum as a raw material by Chinese chemical companies, Japanesechemical companies, and big Chemical companies such as BASF, DuPont, BPchemical etc., but only a small amount of succinic acid for special usesuch as medicine etc. is produced by traditional microbial fermentationmethod. The above-mentioned chemical synthesis methods have a problem ofdischarging large amounts of hazardous waste, effluent, and waste gas(e.g., CO, etc.) generated during a process of producing succinic acid.Particularly, fossil fuels having high possibility of being exhaustedare used as basic material, and thus there is an urgent need to developa method for preparing succinic acids to replace the fossil fuels withalternative fuels such as renewable resources.

To overcome these problems caused by the chemical synthesis process forpreparing succinic acid, studies on producing succinic acids bymicrobial fermentation using various renewable resources have beenintensively and widely conducted by many researchers. Microorganisms,which have been used in succinic acid production, vary, but they can begenerally classified into recombinant Escherichia coli, ruminal bacteria(Actinobacillus, Bacteroides, Mannheimia, Succinimonas, Succinivibrio,etc.) and Anaerobiospirillum (Song et al., Enzyme Microbial Technol.,39:352, 2006).

Among studies on producing succinic acids using recombinant E. coli,there was an attempt to increase succinic acid production by preparing amutant AFP111 (ATCC No. 202021) obtained through a method in which aglucose transport gene (ptsG) is manipulated while genes (ldh and pfl),which are involved in producing lactic acid and formic acid in E. coli,are eliminated, by the University of Chicago research team (U.S. Pat.No. 5,770,435).

The present inventors have amplified a malic enzyme gene (sfcA) involvedin succinic acid production, in recombinant E. coli, NZN111 strain, fromwhich ldh and pfl genes are eliminated, to suppress pyruvic acidaccumulated in the fermentation process of the NZN111 strain, thusincreasing succinic acid production (Hong et al., Biotechnol. Bioeng.,74:89, 2001). Also, a Georgia University-led team of researchers hasconstructed an AFP111/pTrc99A-pyc strain by expressing a pyruvatecarboxylase gene (pyc) in the AFP111 strain, and then used this strainin producing succinic acid (Vemuri et al, J. Ind. Microbiol.Biotechnol., 28:325, 2001). Recently, in order to induce the productionof succinic acid in anaerobic conditions, a Rice University-led team ofresearchers reported that they have constructed recombinant E. colistrains by manipulating genes involved in pathways of Glycolysis, TCAcycle, and Glyoxylate (Lin et al., Eng., 7:116, 2005; Lin et al.,Biotechnol. Bioeng., 90:775, 2005).

Actinobacillus strain and Mannheimia strain, which are a kind of rumenbacteria, and Anaerobiospirillum strain, are known to be excellent inproducing succinic acid, so that studies on the strains have beenactively conducted. Michigan Biotechnology Institute (MBI)-led team ofresearchers in America discovered Actinobacillus succinogenes 130Zstrain (ATCC No. 55618) to develop a method for producing succinic acid,and constructed various mutant strains of Actinobacillus succinogenes,using traditional chemical mutagenesis to use in developing a processfor producing and purifying succinic acid (U.S. Pat. No. 5,521,075; U.S.Pat. No. 5,168,055; U.S. Pat. No. 5,143,834).

However, succinic acid production process using microbial fermentation,developed until now, has a very low productivity of less than 2 g/L/h,and especially it incures a huge cost to separate and purify succinicacid because succinic acid is produced together with large amounts ofvarious organic acids and ethanol as byproducts to some degree duringfermentation. Although the above-mentioned results showed an effect ofdecreasing lactic acid, formic acid, acetic acid, and ethanol asbyproducts in some recombinant strains, they did not show completeelimination of them. In addition, in another recombinant mutant strains,there were some cases where the growth rates of them have become so lowthat overall succinic acid productivity was not increased. Therefore,there is an urgent demand to develop a novel succinic acid-producingstrain, which has a high productivity of succinic acid and prevents theproduction of byproducts (Hong et al., Biotechnol. Lett., 22:871, 2000).

To develop a novel succinic acid-producing strain to satisfy the abovedemands, isolation of a strain having excellent succinic acidproductivity, completion of genome sequence thereof, an understanding ofmetabolic characteristic thereof, and establishing a geneticmanipulation technique required for the construction of a recombinantstrain should be preceded. Up to now, in the case of bacteria havinghigh succinic acid productivity, the full genome sequence of strain M.succiniciproducens MBEL 55E (KCTC 0769BP) was completed, but those ofbacteria such as Actinobacillus, Anaerobiospirillum etc. have not beenreported yet. Although an attempt to try to produce succinic acids byamplifying phosphoenolpyruvate carboxykinase gene (pckA) of A.succinogenes and A. succiniciproducens in E. coli, has been reported(Kim et al., Appl. Environ. Microbiol., 70:1238, 2004; Laivenieks etal., Appl. Environ. Microbiol., 63:2273, 1997), there has been noattempt to try to develop a recombinant succinic acid production strainbased on genome sequence.

The present inventors have reported that they isolated M.succiniciproducens MBEL 55E (KCTC0769BP) producing succinic acid withhigh efficiency from Korean native cattle, and completed genome sequenceand characterized metabolic properties of the strain (Hong et al.,Nature Biotechnol., 22:1275, 2004). Also, the present inventors haveconstructed a bacterial mutant, M. succiniciproducens LPK (KCTC10558BP)by disrupting a gene encoding lactate dehydrogenase (ldhA) and a geneencoding pyruvate formate-lyase (pfl) in M. succiniciproducens MBEL 55E(KCTC 0769BP) which is a kind of rumen bacteria in order to inhibit theproduction of lactic acids and formic acids. In addition to that, thepresent inventions have constructed a mutant M. succiniciproducens LPK7(KCTC1062BP) by disrupting a phosphotransacetylase gene (pta) and anacetate kinase gene (ackA) in the mutant strain, M. succiniciproducensLPK in order to inhibit the production of acetic acid, to culture thebacterial mutants in anaerobic conditions (WO 05/052135 A1; Lee et al.,Appl. Environ. Microbiol. 72:1939, 2006), thus increasing succinic acid.However, in case of such mutant strains, although the production ofbyproducts, formic acid and acetic acid could be suppressed to someextent, a large amount of pyruvic acids were accumulated as a byproductduring fermentation, most of all, the growth rate of the strain hasbecome so low compared with a wild strain that an excellent succinicacid productivity could not be achieved.

Meanwhile, it was reported that a pyruvate formate-lyase gene (pfl)participates in conversion of pyruvic acid into acetyl Coenzyme A(acetyl-CoA), thus affecting cell growth and redistribution of pyruvicacid (Wolfe, Microbial. Mol. Biol. Rev., 69:12, 2005).

Accordingly, the present inventors have made extensive efforts toconstruct a mutant microorganism capable of producing homo-succinicacids at a high yield by minimizing a decrease in microbial growth rateand completely inhibiting the formation of various byproducts includingpyruvic acids and to develop a fermentation method thereof, and as aresult, they have constructed a bacterial mutant M. succiniciproducensPALK (KCTC10973BP) by disrupting a lactate dehydrogenase gene (ldhA), aphosphotransacetylase gene (pta), and an acetate kinase gene (ackA)without disrupting a pyruvate formate-lyase gene (pfl) in M.succiniciproducens MBEL55E (KCTC 0769BP) which is a kind of rumenbacteria, and then fermentated the mutant strain in anaerobic conditionsusing glucose and glycerol as carbon sources, and confirmed that themutant strain can produce nearly homo-succinic acid at a high yield,thereby completing the present invention.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a mutantmicroorganism that has high growth rate and succinic acid productivityand produces only succinic acid at a high yield while producing littleor no other organic acids during the period of anaerobic fermentation.

Another object of the present invention is to provide a method forproducing homo-succinic acid without the accumulation of otherbyproducts, by culturing the mutant microorganism using glucose andglycerol as carbon sources in anaerobic conditions.

To achieve the above objects, the present invention provides a mutantmicroorganism lacking a lactate dehydrogenase gene (ldhA), aphosphotransacetylase gene (pta), and an acetate kinase gene (ackA)while comprising a pyruvate formate-lyase gene (pfl), which has theproperty of producing only succinic acid at a high concentration whileproducing little or no other organic acids in anaerobic conditions,wherein the microorganism is selected from the group consisting of genusMannheimia, genus Actinobacillus and genus Anaerobiospirillum.

Also, the present invention provides a mutant microorganism Mannheimiasucciniciproducens PALK (KCTC10973BP) lacking a lactate dehydrogenasegene (ldhA), a phosphotransacetylase gene (pta), and an acetate kinasegene (ackA) while comprising a pyruvate formate-lyase gene (pfl) in M.succiniciproducens, which has the property of producing succinic acid ata high concentration while producing little or no other organic acids inanaerobic conditions.

Additionally, the present invention provides a method for producing amutant microorganism, the method comprising the steps of: (a) obtaininga mutant microorganism lacking the gene encoding lactate dehydrogenase(ldhA) by disrupting a gene encoding lactate dehydrogenase (ldhA) fromthe genome of succinic acid producing microorganism which is selectedfrom the group consisting of genus Mannheimia, genus Actinobacillus andgenus Anaerobiospirillum, using homologous recombination; and (b)obtaining a mutant microorganism lacking a gene encoding lactatedehydrogenase (ldhA), a gene encoding phosphotransacetylase (pta), and agene encoding acetate kinase (ackA) by disrupting a gene encodingphosphotransacetylase (pta) and a gene encoding acetate kinase (ackA),from the genome of the mutant microorganism lacking the gene encodinglactate dehydrogenase (ldhA), by homologous recombination.

Further, the present invention provides a method for producing succinicacid, the method comprising the steps of: culturing the mutantmicroorganisms in anaerobic conditions; and recovering succinic acidfrom the culture broth.

Another features and embodiments of the present invention will be moreclarified from the following detailed descriptions and the appendedclaims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the succinic acid productionpathway in the mutant microorganism according to the present invention.

FIG. 2 shows a process of constructing a replacement vector fordisrupting ldhA (pMLKO-sacB) by homologous recombination.

FIG. 3 shows a process of constructing a bacterial mutant (M.succiniciproducens LK) by disrupting ldhA gene in Mannheimiasucciniciproducens MBEL55E by homologous recombination.

FIG. 4 shows a process of constructing a replacement vector fordisrupting pta and ackA (pPTA-sacB) by homologous recombination.

FIG. 5 shows a process of constructing a bacterial mutant (M.succiniciproducens PALK) by disrupting pta-ackA genes in M.succiniciproducens LK by homologous recombination.

FIG. 6 is an electrophoresis photograph showing the disruption ofpta-ackA in M. succiniciproducens PALK, wherein M represents 1 kbmarker; lanes 1-6 represent PCR fragments (1.1 kb) using primers, PAU1and SP1; lanes A-F represent PCR fragments (1.5 kb) using primers, SP2and PAD2.

FIG. 7 shows the culture characteristics of the inventive M.succiniciproducens PALK in anaerobic conditions saturated with CO₂.

DETAILED DESCRIPTION OF THE INVENTION, AND Preferred Embodiments

In one aspect, the present invention relates to a mutant microorganismlacking a lactate dehydrogenase gene (ldhA), a phosphotransacetylasegene (pta), and an acetate kinase gene (ackA) while comprising apyruvate formate-lyase gene (pfl), which has the property of producingonly succinic acid at a high concentration while producing little or noother organic acids in anaerobic conditions, wherein the microorganismis selected from the group consisting of genus Mannheimia, genusActinobacillus and genus Anaerobiospirillum.

In the present invention, the succinic acid-producing microorganismrefers to a microorganism capable of producing an excessive large amountof succinic acid compared to the production of ethanol or other organicacids, which can be used for industrial use in succinic acid productionby fermentation.

Typical succinic acid-producing microorganisms include rumen bacteriaand other kind of bacteria producing succinic acids such asAnaerobiospirillum succiniciproducens. From partial genetic information(16s rRNA), enzyme analysis, and fermentation results of Actinobacillussuccinogenes and M. succiniciproducens and which are a kind of rumenbacteria, and Anaerobiospirillum succiniciproducens, and variousbacteria which are known to produce succinic acid until now, it wasfound that main biosynthesis pathway for succinic acid production from acarbon source in succinic acid-producing microorganisms is almostidentical with biosynthesis pathway for succinic acid production inMannheimia sp. which is a kind of rumen bacteria (Table 1; Van der Werfet al, Arch Microbiol., 167:332, 1997; Laivenieks et al., Appl. Environ.Microbiol., 63:2273, 1997; Samuelov et al., App. Environ. Microbiol.,65:2260, 1999; Kim et al., Appl. Environ. Microbiol., 70:1238, 2004).Especially all rumen bacteria and A. succiniciproducens, which areinvolved in the production of succinic acid, convert phosphoenolpyruvateand pyruvate, C3 compounds into oxaloacetate and malate, C4 compoundsusing CO₂-fixing enzyme upon succinic acid production, thus producingsuccinic acid. In addition, rumen bacteria and A. succiniciproducensproduce acetic acid, formic acid, and lactic acid as fermentationbyproducts in anaerobic conditions, thus suggesting that all rumenbacteria, including the Mannheimia sp., and A. succiniciproducens havethe same pathway for succinic acid biosynthesis.

TABLE 1 Succinic acid producing microorganisms Strain ReferencesCytophaga succinicans Anderson et al, J. Bacteriol., 81: 130, 1961Fibrobacter succinogens Wood et al, J. Cereal. Sci., 19: 65, 1994Ruminococcus flavefaciens Iannotti et al., Appl. Environ. Microbiol.,43: 136, 1973 Succinimonas amylolytica Bryant, Bacteriol Rev., 23: 125,1959 Succinivibrio dextrinisolvens Bryant, Bacteriol. Rev., 23: 125,1959 Actinobacillus succinogenes Clutter et al., Int. J. Syst.Bacteriol., 49: 207, 1999 Mannheimia succiniciproducens Hong et al.,Nature Biotechnol., 22: 1275, 2004

In the present invention, a succinic acid-producing mutant microorganismwith a high growth rate, which is capable of producing succinic acid ata high concentration while producing little or no other organic acids,was constructed by manipulating the genome of a succinic acid-producingmicroorganism. In other words, a lactate dehydrogenase gene (ldhA), aphosphotransacetylase gene (pta), and an acetate kinase gene (ackA) weredisrupted in the genome DNA of M. succiniciproducens MBEL55E(KCTC0769BP), thereby constructing a bacterial mutant M.succiniciproducens PALK (KCTC 10973BP) which shows a high growth rateand produces succinic acid at a high concentration while producinglittle or no other organic acids.

In the present invention, for the disruption of each gene, a method ofsubstituting the genes by homologous recombination to inactivate wasused, but any method can be used without limitations as long as it isgenetic manipulation method where the corresponding gene can be modifiedor eliminated, such that an enzyme encoded by the corresponding gene isnot generated.

In the present invention, the said succinic acid-producingmicroorganisms are Anaerobiospirillum sp. bacteria or rumen bacteriawhich are selected from the group consisting of Mannheimia sp. andActinobacillus sp.

In the present invention, the mutant microorganism is preferably ahomogeneous fermentation strain producing only succinic acid whileforming little or no other organic acids as byproducts, the each amountsof other organic acids produced is preferably less than 1 wt % based onthe amount of succinic acid produced, and the other organic acids arepreferably any one or more organic acids selected from the groupconsisting of lactic acid, acetic acid, formic acid, and pyruvic acid.

In another aspect, the present invention relates to a rumen bacterialmutant M. succiniciproducens PALK (KCTC10973BP), lacking a lactatedehydrogenase gene (ldhA), a phosphotransacetylase gene (pta), and anacetate kinase gene (ackA) in M. succiniciproducens while a pyruvateformate-lyase gene (pfl) is maintained in M. succiniciproducens, whichhas the property of producing succinic acid at a high concentrationwhile producing little or no other organic acids in anaerobicconditions.

The bacterial mutant M. succiniciproducens LPK7 (KCTC10626BP), which wasconstructed by disrupting a gene encoding lactate dehydrogenase (ldhA),a gene encoding pyruvate formate-lyase (pfl), a gene encodingphosphotransacetylase (pta), and a gene encoding acetate kinase (ackA)in the genome of Mannheimia sp., was used in order to compare thesuccinic acid productivity and growth rate of M. succiniciproducens PALK(KCTC10973BP) according to the present invention with those oftraditional bacterial mutants. Herein, the bacterial mutant M.succiniciproducens LPK7 (KCTC10626BP) has an additional disruption of agene encoding pyruvate formate-lyase (pfl) in the mutant strain M.succiniciproducens PALK (KCTC10973BP) according to the present invention(WO2005/052135; Lee et al., Appl. Environ. Microbiol. 72:1939, 2006).

M. succiniciproducens PALK (KCTC10973BP) according to the presentinvention has a high succinic acid productivity and produces little orno byproducts such as lactic acid, acetic acid, formic acid, and pyruvicacid, thus showing excellent properties compared to wild-type M.succiniciproducens MBEL55E (KCTC0769BP) for succinic acid production andpreviously constructed bacterial mutant M. succiniciproducens LPK7(KCTC10626BP), and can produce succinic acid at a high yields due to ahigh growth rate thereof, high succinic acid productivity andhomo-succinic acid production by preventing pyruvic acid accumulation,compared to previously constructed bacterial mutant M.succiniciproducens LPK7 (KCTC10626BP).

In still another aspect, the present invention relates to a method forconstructing a mutant microorganism, the method comprising the steps of:(a) obtaining a mutant microorganism lacking a gene encoding lactatedehydrogenase (ldhA) by disrupting a gene encoding lactate dehydrogenase(ldhA) in genome of succinic acid producing microorganism which isselected from the group consisting of genus Mannheimia, genusActinobacillus and genus Anaerobiospirillum, using homologousrecombination; and (b) obtaining a mutant microorganism lacking a geneencoding lactate dehydrogenase (ldhA), a gene encodingphosphotransacetylase (pta), and a gene encoding acetate kinase (ackA)by disrupting a gene encoding phosphotransacetylase (pta) and a geneencoding acetate kinase (ackA) in genome of the mutant microorganismlacking a gene encoding lactate dehydrogenase (ldhA) using homologousrecombination.

In the present invention, the homologous recombination of the step (a)is preferably performed using a genetic exchange vector containing adisrupted ldhA, and the homologous recombination of the step (b) ispreferably performed using a genetic exchange vector containing adisrupted pta-ackA.

In the present invention, the genetic exchange vector containing adisrupted ldhA is preferably pMLKO-sacB, and the genetic exchange vectorcontaining a disrupted pta-ackA is preferably pMPKO-sacB.

The present invention also provides a method for producing succinicacid, the method comprising the steps of: culturing the above-mentionedmutant microorganism in anaerobic conditions; and recovering succinicacid from the culture broth.

In the present invention, for the culture, glucose or glycerol ispreferably used as a carbon source, and the each amount of other organicacids produced as byproducts is preferably less than 1 wt % based on theamount of succinic acid produced.

In yet another aspect, the present invention relates to a method forpreparing succinic acid, the method comprising the steps of: culturingthe mutant microorganism in anaerobic conditions; and recoveringsuccinic acid from the culture broth.

The culture of the succinic acid producing mutant microorganismaccording to the present invention and recovery process of succinic acidcan be performed by the culture methods known in the conventionalfermentation process and methods for separating and purifying succinicacid.

In the present invention, for the culture, glucose or glycerol ispreferably used as a carbon source, and the each amounts of otherorganic acids produced as byproducts is preferably less than 1 wt %based on the amount of succinic acid produced.

EXAMPLES

The present invention will hereinafter be described in further detailsby examples. It will however be obvious to a person skilled in the artthat these examples are given for illustrative purpose only, and thepresent invention is not limited to or by the examples.

Particularly, the following examples illustrate only Mannheimia sp.which is a succinic acid-producing microorganism as a host cell in orderto delete the genes according to the present invention. However, it isobvious to a person skilled in the art that a mutant microorganismproducing homo-succinic acid can be obtained even when other kinds ofsuccinic acid producing microorganisms are used.

Example 1 Construction of ldhA Disruption Vector (pMLKO-sacB)

In order to disrupt a lactate dehydrogenase gene (ldhA) in the genome ofa succinic acid-producing microorganism by homologous recombination, agene exchange vector was constructed in the following manner. First, thegenomic DNA of M. succiniciproducens MBEL55E (KCTC 0769BP), as atemplate, was subjected to PCR using primers set forth in SEQ ID NO: 1and SEQ ID NO: 2 below, and then, the obtained PCR fragment containingldhA-homologous region 1 (L1) was cut with SacI and PstI and introducedinto pUC18 vector (New England Biolabs, Inc., USA), thereby constructingpUC18-L1.

SEQ ID NO: 1: 5′-CAGTGAAGGAGCTCCGTAACGCATCCGCCG SEQ ID NO: 2:5′-CTTTATCGAATCTGCAGGCGGTTTCCAAAA

In addition, the genomic DNA of M. succiniciproducens MBEL55E (KCTC0769BP), as a template, was subjected to PCR using primers set forth inSEQ ID NO: 3 and SEQ ID NO: 4 below, and then, the obtained PCR fragmentcontaining ldhA-homologous region 2 (L2) was cut with PstI and HindIIIand introduced into the pUC18-L1, thereby constructing pUC18-L1-L2.

SEQ ID NO: 3: 5′-GTACTGTAAACTGCAGCTTTCATAGTTAGC SEQ ID NO: 4:5′-GCCGAAAGTCAAGCTTGCCGTCGTTTAGTG

In order to insert kanamycin-resistant gene as a selection marker in thepUC18-L1-L2, pUC4K vector (Pharmacia, Germany) was cut with PstI, andthe resulting kanamycin-resistant gene was fused with pUC18-L1-L2 cutwith PstI, thereby constructing pUC18-L1-KmR-L2. A linker set forth inSEQ ID NO: 5 was inserted into the pUC18-L1-KmR-L2 cut with SacI,thereby making a new XbaI cutting site.

SEQ ID NO: 5: 5′-TCTAGAAGCT

In order to insert sacB gene in the pUC18-L1-KmR-L2 into which the XbaIcutting site has been inserted, PCR on pKmobsacB (Schafer et al., Gene,145:69, 1994) as a template was performed using primers set forth in SEQID NO: 6 and 7. Then the resulting PCR fragment containing sacB gene wascut with XbaI, and inserted into the new XbaI restriction enzyme site ofpUC18-L1-KmR-L2 into which the above XbaI cutting site has beeninserted, thereby constructing pMLKO-sacB, an exchange vector fordisrupting ldhA gene (FIG. 2).

SEQ ID NO: 6: 5′-GCTCTAGACCTTCTATCGCCTTCTTGACG SEQ ID NO: 7:5′-GCTCTAGAGGCTACAAAATCACGGGCGTC

Example 2 Construction of M. succiniciproducens LK Strain

A mutant strain was constructed by disrupting ldhA gene in the genome ofM. succiniciproducens MBEL55E (KCTC0769BP) using pMLKO-sacB constructedin Example 1 as a genetic exchange vector for disrupting ldhA gene (FIG.3).

In other words, M. succiniciproducens MBEL55E (KCTC0769BP) was plated onLB-glucose agar medium containing 10 g/L of glucose, and cultured at 37°C. for 36 hours. The colony formed was inoculated in 10 ml of LB-glucoseliquid medium, and cultured for 12 hours. 1% of the culture broth, whichhad been sufficiently grown, was inoculated in 100 ml of LB-glucoseliquid medium, and cultured in a shaking incubator at 200 rpm and 37° C.

When the culture broth reached an OD₆₀₀ of about 0.3-0.4 after 4˜5hours, it was centrifuged at 4° C. and 4,500 rpm for 20 minutes tocollect cells. Then, the cells were resuspended in 200 ml of 10%glycerol solution at 4° C. The suspension was centrifuged at 4° C. and5,500 rpm for 20 minutes, and the cells were collected. Afterresuspending and collecting in one-half of the above glycerol solutiontwice in the same manner as the above-described processes, the cellswere suspended in glycerol at a volume ratio of 1:1, to obtain cellconcentrate.

The cell concentrate thus obtained was mixed with the genetic exchangevector pMLKO-sacB constructed in Example 1, and then pMLKO-sacB wasintroduced into the cultured M. succiniciproducens MBEL55E (KCTC0769BP)by electroporation under conditions of 1.8 kV, 25 μF and 200 ohms. 1 mlof LB-glucose liquid medium was added to the pMLKO-sacB-introducedstrain, and precultured in a shaking incubator at 37° C. and 200 rpm forone hour. The culture broth was plated on LB-glucose solid mediumcontaining an antibiotic kanamycin (final concentration of 25 μg/ml) andcultured at 37° C. for 48 hours or more. In order to select a colonywhere only double crossover occurred, the colonies formed were streakedon LB-sucrose solid medium containing kanamycin (25 μg/ml) and 100 g/Lsucrose. After 24 hours, the formed colonies were streaked again on thesame medium.

The colonise (mutant) formed on the medium were cultured in LB-glucoseliquid medium containing an antibiotic, and genomic DNA was isolatedfrom the cultured strain by the method described in Rochelle et al.(Rochelle et al., FEMS Microbiol. Lett., 100:59, 1992). PCR wasperformed using the isolated mutant genomic DNA as a template, and thePCR product was electrophoresed to confirm the disruption of ldhA genein the genomic DNA.

In order to confirm the disruption of the ldhA gene, PCRs were performedtwice in the following manners. First, the mutant genomic DNA as atemplate was subjected to PCR using primers set forth in SEQ ID NO: 8and SEQ ID NO: 9.

SEQ ID NO: 8: 5′-GACGTTTCCCGTTGAATATGGC (KM1) SEQ ID NO: 9:5′-CATTGAGGCGTATTATCAGGAAAC (LU1)

Then, the mutant genomic DNA as a template was subjected to PCR usingprimers set forth in SEQ ID NO: 10 and SEQ ID NO: 11.

SEQ ID NO: 10: 5′-GCAGTTTCATTTGATGCTCGATG (KM2) SEQ ID NO: 11:5′-CCTCTTACGATGACGCATCTTTCC (LD2)

The PCR fragments obtained in the two PCRs were subjected to gelelectrophoresis to confirm the disruption of ldhA by their size. The PCRfragments of the genomic DNA having disrupted ldhA were confirmed by thefact that the product resulted from the PCR using the primers of SEQ IDNO: 8 (KM1) and SEQ ID NO: 9 (LU1) has a size of 1.5 kb, and at the sametime the product resulted from the PCR using the primers of SEQ ID NO:10 (KM2) and SEQ ID NO: 11 (LU2) has a size of 1.7 kb. The position ofeach primer is shown in FIG. 3.

The mutant strain M. succiniciproducens LK was constructed by disruptingldhA gene in the genome of M. succiniciproducens MBEL55E according tothe above method.

Example 3 Construction of Gene Exchange Vector (pPTA-sacB) for theDisruption of pta and ackA

In order to disrupt pta and ackA in the genome of M. succiniciproducensLK strain by homologous recombination, a genetic exchange vector wasconstructed in the following manner. A vector pKmobsacB containing asacB gene, as a template, was subjected to PCR using primers set forthin SEQ ID NO: 12 and SEQ ID NO: 13. The resulting sacB product was cutwith PstI and BamHI and inserted into pUC19 (Stratagene Cloning Systems.USA), thereby constructing pUC19-sacB (FIG. 4).

SEQ ID NO: 12: 5′-AGCGGATCCCCTTCTATCGCCTTCTTGACG SEQ ID NO: 13:5′-GTCCTGCAGGGCTACAAAATCACGGGCGTC

Meanwhile, the genomic DNA of M. succiniciproducens LK as a template wassubjected to PCR using primers set forth in SEQ ID NO: 14 and SEQ ID NO:15, and the resulting PCR fragment containing pta-ackA homologous region1 was cut with XbaI and BamHI. In addition, the genomic DNA of M.succiniciproducens LK as a template was subjected to PCR using primersset forth in SEQ ID NO: 16 and SEQ ID NO: 17, and the resulting PCRfragment containing pta-ackA homologous region 2 was cut with XbaI andSacI. Then, these fragments were inserted into BamHI and SacI site ofpUC19, thereby constructing pUC19-PTA12.

SEQ ID NO: 14: 5′-GCTCTAGATATCCGCAGTATCACTTTCTGCGC SEQ ID NO: 15:5′-TCCGCAGTCGGATCCGGGTTAACCGCACAG SEQ ID NO: 16:5′-GGGGAGCTCGCTAACTTAGCTTCTAAAGGCCATGTTTCC SEQ ID NO: 17:5′-GCTCTAGATATCCGGGTCAATATCGCCGCAAC

In order to insert a spectinomycin-resistant gene (GenBank X02588; SpR)as a selective marker in pUC19-PTA12, plasmid pIC156 (Steinmetz et al,Gene, 142:79, 1994) containing a spectinomycin-resistant gene (GenBankX02588), as a template, was subjected to PCR using primers set forth inSEQ ID NO: 18 and SEQ ID NO: 19, and the resulting PCR fragmentcontaining SpR gene was cut with EcoRV and introduced into thepUC19-PTA12, thereby constructing pUC19-PTA1S2 having thespectinomycin-resistant gene. The constructed pUC19-PTA1S2 was cut withSacI and BamHI and introduced into the above constructed pUC19-SacB,thereby constructing an exchange vector (pPTA-sacB) for the disruptionof pta and ackA (FIG. 4).

SEQ ID NO: 18: 5′-GAATTCGAGCTCGCCCGGGGATCGATCCTC SEQ ID NO: 19:5′-CCCGGGCCGACAGGCTTTGAAGCATGCAAATGTCAC

Example 4 Construction of M. succiniciproducens PALK Strain

A mutant strain was constructed by disrupting pta and ackA gene in thegenome of M. succiniciproducens LK using pPTA-sacB, an exchange vectorfor the disruption of phosphotransacetylase gene (pta) and acetatekinase gene (ackA) constructed in Example 3 (FIG. 4).

In other words, M. succiniciproducens LK constructed in Example 2 wasplated on LB-glucose agar medium containing 10 g/L of glucose, andcultured at 37° C. for 36 hours. The colony formed was inoculated in 10ml of LB-glucose liquid medium, and cultured for 12 hours. 1% of theculture broth, which had been sufficiently grown, was inoculated in 100ml of LB-glucose liquid medium, and cultured in a shaking incubator at200 rpm and 37° C.

Cell concentrate was collected from the resulting culture broth in thesame manner as described in Example 2. The collected cell concentratewas mixed with the genetic exchange vector pPTA-sacB constructed inExamples 3, and then pPTA-sacB was introduced into the M.succiniciproducens LK by electroporation under conditions of 2.5 kV, 50μF and 200 ohms. 800 ml of LB-glucose liquid medium was added to thepPTA-sacB-introduced strain, and precultured in a thermostat at 37° C.for one and a half hour. In order to induce a double crossover, theculture broth was plated on TSB-sucrose solid medium (Tryptic Soy Broth(Becton, Dickinson and Company) solid medium containing 100 g/L ofsucrose) containing an antibiotic spectinomycin (final concentration of50 μg/ml) and cultured at 37° C. for 48 hours or more. In order toscreening a colony where only double crossover occurred, the coloniesformed were streaked on TSB-sucrose medium containing 50 μg/mlspectinomycin and TSB agar medium containing 50 μg/ml ampicillin,respectively, and cultured at 37° C. for 12 hours. Then, the colonies,which were formed on the TSB-sucrose medium containing 50 μg/mlspectinomycin but not formed on the TSB medium containing 50 μg/mlampicillin, were selected and streaked again on the TSB-sucrose mediumcontaining 50 μg/ml spectinomycin. The colonies formed herein werescreened again using the spectinomycin-containing medium and theampicillin-containing medium, and then the colonies which showedrequired results were selected ultimately. The isolated mutant genomicDNA as a template was amplified by PCR and the PCR product waselectrophoresed to confirm the disruption of pta-ackA.

To confirm the disruption of pta-ackA, PCRs were performed twice in thefollowing manner. First, the mutant genomic DNA as a template wassubjected to PCR using primers set forth in SEQ ID NO: 20 and SEQ ID NO:21. Then, the mutant genomic DNA as a template was subjected to PCRusing primers set forth in SEQ ID NO: 22 and SEQ ID NO: 23.

SEQ ID NO: 20: 5′-CCTGCAGGCATGCAAGCTTGGGCTGCAGGTCGACTC (SP1) SEQ ID NO:21: 5′-GCTGCCAAACAACCGAAAATACCGCAATAAACGGC (PAU1) SEQ ID NO: 22:5′-GCATGTAACTTTACTGGATATAGCTAGAAAAGGCATCGGGGAG (SP2) SEQ ID NO: 23:5′-GCAACGCGAGGGTCAATACCGAAGGATTTCGCCG (PAD2)

The products obtained in the two PCRs were subjected to gelelectrophoresis to confirm the disruption of pta-ackA by their size(FIG. 6). The disruption of pta-ackA was confirmed by the fact theproduct resulted from the PCR using the primers of SEQ ID NO: 20 and SEQID NO: 21 (SP1 primer and PAU1 primer) has a size of 1.1 kb, and at thesame time the product resulted from the PCR using the primers of SEQ IDNO: 22 and SEQ ID NO: 23 (SP2 primer and PAD2 primer) has a size of 1.5kb. The position of each primer is shown in FIG. 5.

The mutant strain constructed by the disruption of pta-ackA from thegenome of M. succiniciproducens LK as the above-described method, i.e.,a mutant strain resulted from the disruption of ldhA, pta and ackA fromthe genome of M. succiniciproducens, was named “M. succiniciproducensPALK” and deposited under accession number “KCTC10973BP” on Jul. 26,2006 in the Korean Collection for Type Cultures (KCTC; 52, Eoeun-dong,Yuseong-gu, Daejeon-si, Republic of Korea), Korean Research Institute ofBioscience and Biotechnology, which is an international depositaryauthority.

Example 5 Production of Homo-Succinic Acid Using M. succiniciproducensPALK

M. succiniciproducens PALK (KCTC10973BP) constructed in Example 4 wasplated on 10 ml of composition medium containing 5 g/L of glucose, andcultured in anaerobic conditions at 39° C. for 8 hours, and then againmoved to 250 ml of composition medium containing 5 g/L of glucose andcultured at 39° C. At this time 50 μg/ml spectinomycin was added to themedium as an antibiotic. 250 ml of the culture broth of M.succiniciproducens PALK was inoculated in the bioreactor containing 2.25L of composition medium (1 g/L of NaCl, 2 g/L of (NH₄)₂HPO₄, 0.02 g/L ofCaCl₂.2H₂O, 0.2 g/L of MgCl₂.6H₂O, 8.709 g/L of K₂HPO₄, 0.5 g/L ofcystein, 0.5 g/L of methionine, 0.5 g/L of alanine, 0.5 g/L ofasparagines, 0.5 g/L of aspartic acid, 0.5 g/L of praline, 0.5 g/L ofserine, 0.005 g/L of nicotinic acid, 0.005 g/L of Ca-pantothenate, 0.005g/L of pyridoxine-HCl, 0.005 g/L of thiamine, 0.005 g/L of ascorbicacid, and 0.005 g/L of biotin), and the fermentation was performed underconditions of a first glucose concentration of 18.2 g/L (100 mM), afirst glycerol concentration of 9.2 g/L (100 mM) at 39° C. During thefermentation, the pH of the culture was adjusted to 6.5 by using ammoniawater, and 50 μg/ml spectinomycin was added to the medium as anantibiotic. In order to produce succinic acid at a high concentration,whenever glucose was completely exhausted, the glucose concentration ofthe culture broth was adjusted to about 18.2 g/L (100 mM) by addingconcentrated glucose solution.

M. succiniciproducens MBEL55E (KCTC0769BP) and the succinicacid-producing mutant strain M. succiniciproducens LPK7 (KCTC10626BP)were fermented to produce succinic acid in the same manner as describedabove.

The concentration of cells in the culture broth was measured with aspectrophotometer, and then calculated using the previously measuredlight absorption of spectrophotometer and the verification test fordried-cell weight. During the fermentation, samples were collected fromthe bioreactor regularly. The collected samples were centrifuged at13,000 rpm for 10 minutes, and then the supernatants were used toanalyze the concentrations of organic acids, glucose, and glycerol byusing a High-Performance Liquid Chromatography.

As a result, when M. succiniciproducens PALK (KCTC10973BP) of thepresent invention was compared with M. succiniciproducens MBEL55E(KCTC0769BP) and M. succiniciproducens LPK7 (KCTC10626BP), it showedhigher succinic acid productivity while producing little or no otherorganic acids as byproducts than M. succiniciproducens MBEL55E(KCTC0769BP) and M. succiniciproducens LPK7 (KCTC10626BP) as shown inFIG. 7 and Table 2. Although 0.45 g/L of acetic acid and 0.24 g/L ofpyruvic acid were detected, it is obvious to a person skilled in the artthat these amount are the minimum amounts of organic acids produced fora strain to grow and can be ignored because they were produced inamounts of less than 1 wt % compared to produced succinic acid.

TABLE 2 Succinic acid-productivity of M. succiniciproducens PALK(KCTC10973BP) MBEL55E LPK7 PALK Strain (KCTC0769BP) (KCTC10626BP)(KCTC10973BP) Final concentration of 10.49 13.40 45.79 succinic acid(g/L) Glucose consumption 22.50 19.98 53.00 (g/L) Succinic acid yield (g0.47 0.67 0.86 succinic acid/g glucose) Increasing rate of — 42.55 82.98succinic acid compared to wild strain (MBEL55E) (%) Final concentrationof 4.96 0.53 0.45 acetic acid (g/L) Final concentration of 3.47 0.270.00 lactic acid (g/L) Final concentration of 0.00 2.47 0.24 pyruvicacid (g/L) Cell specific growth rate 0.81 0.30 0.69 (1/h)

INDUSTRIAL APPLICABILITY

As described and provided above in detail, the present inventionprovides the succinic acid-producing mutant microorganism having a highgrowth rate, which produces succinic acid at a high concentration whileproducing little or no other organic acids during the culture inanaerobic conditions, and the method for producing succinic acid usingthe same. The inventive mutant microorganism has the ability of having ahigh growth rate and succinic acid productivity while producing littleor no organic acids, as compared to the prior strains producing succinicacid. Thus, the inventive mutant microorganism is useful to producesuccinic acid for industrial use.

While the present invention has been described in detail with referenceto the specific features, it will be apparent to persons skilled in theart that this description is only for a preferred embodiment and doesnot limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

SEQUENCE LISTING

The electronic file was attached.

What is claimed is:
 1. A mutant rumen bacterium, said mutant rumenbacterium lacking (i) a lactate dehydrogenase gene (ldhA), (ii) aphosphotransacetylase gene (pta), and (iii) an acetate kinase gene(ackA), said mutant rumen bacterium comprising non-disrupted pyruvateformate-lyase gene (pfl), and said mutant rumen bacterium having theproperty of producing succinic acid in anaerobic conditions, whereinsaid rumen bacterium is selected from the group consisting of genusMannheimia, genus Actinobacillus and genus Anaerobiospirillum, andwherein the amount of any other organic acid produced by the mutantrumen bacterium as a byproduct in the production of succinic acid by themutant rumen bacterium is less than 1 wt % based on the weight ofsuccinic acid that is produced.
 2. The mutant rumen bacterium accordingto claim 1, wherein said any other organic acid is selected from thegroup consisting of lactic acid, acetic acid, formic acid, and pyruvicacid.
 3. A mutant rumen bacterium, Mannheimia succiniciproducens PALK(KCTC10973BP).
 4. A method for producing the mutant rumen bacterium ofclaim 1, the method comprising the steps of: (a) obtaining a mutantrumen bacterium lacking a gene encoding lactate dehydrogenase (ldhA) bydisrupting a gene encoding lactate dehydrogenase (ldhA) in the genome ofa succinic acid producing rumen bacterium which is selected from thegroup consisting of genus Mannheimia, genus Actinobacillus and genusAnaerobiospirillum, using homologous recombination; and (b) obtaining amutant rumen bacterium lacking a gene encoding lactate dehydrogenase(ldhA), lacking a gene encoding phosphotransacetylase (pta), and lackinga gene encoding acetate kinase (ackA), by disrupting a gene encodingphosphotransacetylase (pta), and disrupting a gene encoding acetatekinase (ackA), without disrupting a gene encoding pyruvate formate lyase(pfl), in the genome of the mutant rumen bacterium lacking a geneencoding lactate dehydrogenase (ldhA), using homologous recombination.5. The method for producing the mutant rumen bacterium according toclaim 4, wherein the homologous recombination of the step (a) isperformed using a genetic exchange vector containing a disrupted ldhA.6. The method for producing the mutant rumen bacterium according toclaim 4, wherein the homologous recombination of the step (b) isperformed using a genetic exchange vector containing a disruptedpta-ackA.
 7. The method for producing the mutant rumen bacteriumaccording to claim 6, wherein the genetic exchange vector containing adisrupted ldhA is pMLKO-sacB.
 8. The method for producing the mutantrumen bacterium according to claim 6, wherein the genetic exchangevector containing a disrupted pta-ackA is pMPKO-sacB.
 9. A method forproducing succinic acid, the method comprising the steps of: culturingthe mutant rumen bacterium of claim 1 in a culture broth under anaerobicconditions; and recovering succinic acid from the culture broth.
 10. Themethod for producing succinic acid according to claim 9, wherein glucoseor glycerol is used as a carbon source for the culture.
 11. The methodfor preparing succinic acid according to claim 9, wherein the amount ofany other organic acid produced by the mutant rumen bacterium as abyproduct in the production of succinic acid by the mutant rumenbacterium is less than 1 wt % based on the weight of succinic acid thatis produced.
 12. A method for preparing succinic acid, the methodcomprising the steps of: culturing the mutant microorganism Mannheimiasucciniciproducens PALK (KCTC10973BP) in a culture broth under anaerobicconditions; and recovering succinic acid from the culture broth.
 13. Themethod for preparing succinic acid according to claim 12, whereinglucose or glycerol is used as a carbon source for the culture.
 14. Themethod for preparing succinic acid according to claim 12, wherein theamount of any other organic acid produced as a byproduct in theproduction of succinic acid by the mutant rumen bacterium is less than 1wt % based on the weight of succinic acid that is produced.